RU2740007C1 - Method for simulating diet-induced metabolic syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to experimental medicine. Method for simulating diet-induced metabolic syndrome in laboratory animals involves using a high-fat and high-carbohydrate diet, characterized in that animals receive ration for 12 weeks, containing standard fodder (specific weight 66 %) with addition of animal fat (melted lard, specific weight 17 %), fructose (specific weight 17 %), cholesterol (specific gravity 0.1 %), and consuming 20 % solution of fructose instead of drinking water.

EFFECT: technical result is a model of metabolic syndrome, adequately reflecting the main features of metabolic disorders developing in the given syndrome.

1 cl, 5 dwg

Description

The invention relates to the field of experimental medicine, namely to a method for modeling metabolic syndrome using a high-fat, high-carbohydrate diet, and can be used in medicine and clinical pharmacology for the search and development of drugs intended for the prevention and treatment of metabolic and hemodynamic disorders developing in metabolic syndrome.

A known method for modeling metabolic syndrome in rats using a diet high in fat and high in sugar with the addition of corn oil [1]. The disadvantage of this method is that this method does not give an idea about the degree of development of dyslipidemia in animals, which is an important criterion in the diagnosis of metabolic syndrome, and also involves the intragastric administration of corn oil, which requires special technical skills from the researcher.

There is also known a method of obtaining a model of metabolic syndrome in rats using high-fat feed (60 parts of standard feed, 8 parts of egg powder, 12 parts of lard, 12 parts of sucrose, 6 parts of peanut powder, 1 part of milk powder, 1 part of cholesterol, 0.01 part bile salts, 3 parts palm oil and 3 parts coconut oil.) for 18 weeks [2]. The disadvantage of this method is that it does not allow judging the development of impaired glucose tolerance and insulin resistance in animals, as important symptoms of metabolic syndrome, and the composition of the diet is complex and costly to reproduce.

A known method for modeling metabolic syndrome in rodents based on the use of purified feed with a high fat content (20.0-25.0 parts of casein, 8.0-10.0 parts of starch, 18.0-20.0 parts of sucrose, 2.0 -3.0 parts of vegetable oil, 18.0-22.0 parts of animal fat, 5.0-6.0 parts of cellulose, 0.2-0.5 parts of L-cystine, 1.0-2.0 parts of dicalcium phosphate , 0.5-1.0 parts of calcium carbonate, 1.5-2.0 parts of potassium citrate, 0.15-0.25 parts of choline bitartrate 1.0-2.0 parts of complex minerals, 1.0-1, 5 parts of the vitamin complex and 10.0-12.0 parts of maltodextrin [3] Disadvantages - difficult to reproduce multicomponent diet.

A known method of obtaining a model of metabolic syndrome in mice using a diet that contains NO2- and NO3- ions [4]. This diet results in visceral fat accumulation, hyperlipidemia, impaired glucose tolerance and insulin resistance without weight gain in animals. Disadvantages - this model of metabolic syndrome is accompanied by the accumulation of nitrates in the body of mice, which not only reproduce the signs of the syndrome under study, but also have a general toxic effect.

The closest to the claimed technical solution in terms of the technical essence and the achieved result is a method for modeling metabolic syndrome in Sprague-Dawley rats using a diet high in fat and high in sugar with the addition of corn oil for 90 days [5]. The described method is taken as a prototype of the invention. This method of modeling metabolic syndrome reproduces the main signs of metabolic syndrome, such as obesity, insulin resistance, increased glucose tolerance, and the development of non-alcoholic fatty liver disease.

The disadvantages of this method is the lack of a functional assessment of the degree of development of dyslipidemia in animals, and the need for special practical skills for the introduction of diet components.

The technical objective of the invention is to create an accessible and easily reproducible model of diet-induced metabolic syndrome based on the use of a combined high-fat and high-carbohydrate diet.

The problem is solved by the fact that the modeling of the metabolic syndrome in laboratory animals is performed within 12 weeks using a specially developed high-fat and high-carbohydrate diet, including standard feed (specific gravity 66%) with the addition of animal fat (rendered lard, specific gravity 17%), fructose (specific gravity 17%), cholesterol (specific gravity 0.1%), as well as replacing drinking water with a 20% fructose solution, followed by an assessment of the severity of metabolic disorders reflected in the development of obesity, dyslipidemia, hyperglycemia, impaired glucose tolerance, insulin resistance, fatty degeneration of the liver, increased blood pressure.

New in the proposed method is the combined inclusion in the diet of animals of a large amount of animal fats and fructose as a carbohydrate, as well as the addition of cholesterol, which induce the development of metabolic syndrome. The proposed high-calorie diet is as close as possible to the diet of a modern person, its use in laboratory animals can repeat the pathogenetic factors and phenomenology of disorders that occur in people with metabolic syndrome.

The advantage of the method proposed as the invention is that the diet used to simulate the metabolic syndrome is easily reproducible, available in terms of its component composition and does not require special skills for its implementation; also against the background of the use of this combined high-fat diet with the addition of fructose, animals develop hyperglycemia, a persistent increase blood pressure,

triglyceridemia, obesity, signs of atherogenesis and fatty degeneration of the liver.

The proposed method for modeling metabolic syndrome most fully reveals the clinical picture of disorders inherent in metabolic syndrome and can be used to create approaches to its prevention and therapy.

The essence of the invention boils down to the fact that the experimental metabolic syndrome is caused in laboratory animals by a 12-week high-fat and high-carbohydrate diet, based on a standard feed with the addition of ghee (17%), fructose (17%) and cholesterol (0.1%) together with a 20% fructose solution instead of drinking water. At the beginning of the study and after 4, 8, 12 weeks, the animals are measured for body weight and blood pressure. In the last week of the study, animals undergo glucose tolerance and insulin tolerance tests, and blood is taken for biochemical studies of lipid (triglycerides, cholesterol, high density lipoprotein) and protein (urea) metabolism parameters. At the end of the experiment, the animals are sacrificed by CO2 asphyxia. By the method of dissection, the aorta, liver and visceral adipose tissue are isolated, the specific gravity of the liver and fat, as well as the content of triglycerides and cholesterol in the liver and aorta are determined. The results obtained are analyzed by methods of statistical data processing.

The method is carried out as follows.

The studies are performed on male Wistar rats (at least 20 _; animals weighing from 200 to 250 g, age at the beginning of the experiment 6 weeks) in compliance with the principles of humanity set forth in the directives of the European Community (86/609 / EEC) and the Declaration of Helsinki. Animals are kept in standard vivarium conditions with 12 hours of daylight hours and free access to food and water. The rats are randomly assigned to a control group and an experimental group. Rats of the control group are kept on a standard diet (food "Delta Feeds", Biopro, RF, total caloric content of 300 kcal / 100 g, fats account for 18% of the total caloric content). The composition of the feed: wheat, barley, bran, corn gluten, fish flour, protein feed mixture, sunflower oil, soybean meal [6].

For rats of the experimental group, a diet is prepared, which includes the above-described Delta Feeds feed (specific gravity 66%), animal fat (melted pork lard, lard, specific gravity 17%), fructose (specific gravity 17%), cholesterol (specific gravity weight 0.1%). The products are ground in a blender into a homogeneous mixture, granules up to 11 mm in diameter are formed from the resulting mass and dried in an oven at 25 ° C. The finished food is stored in a refrigerator at 4 ° C for no more than 3 days. Drinking water is replaced with a 20% fructose solution, which is prepared just before serving the animals daily. The calorie content of the feed is 360 kcal / 100 g of food (fat accounts for 54% of the total calorie content), also an additional 80 kcal / 100 g due to a 20% fructose solution.

Before the beginning and after 4, 8, 12 weeks of the experiment, the animals are measured for body weight, weighing on an analytical balance, and blood pressure (using a non-invasive measurement system using the tail cuff "Systola", Neurobotics, RF). Food and water intake by rats is monitored weekly. In the last week of the experiment, glucose tolerance and insulin tolerance tests are performed [7,8]. The tests are performed in the morning on an empty stomach after depriving rats of food for 12 hours and replacing the fructose solution in the experimental group with ordinary drinking water. For the glucose tolerance test, the rats of the control and experimental groups are intragastrically injected with a probe using a glucose solution at a dose of 2 g / kg (D-glucose, Sigma-Aldrich, USA). For the insulin-tolerant test, animals are injected subcutaneously with short-acting insulin at a dose of 0.75 IU / kg (NovoRapid Penfil, Novo Nordisk A / S, Denmark). Then from the tail vein of the rats after 15, 30, 60, 90 and 120 minutes, blood is taken in a volume of 10 μl for subsequent determination of the glucose concentration in the blood. The glucose content is determined by the enzymatic method using a set of reagents ("Glucose-Novo", Vector-West, RF) spectrophotometrically (spectrophotometer SF-2000, RF). It is necessary to maintain an interval of at least 3 days between tests. To assess insulin resistance, the rate constant of glucose utilization is calculated according to the data of the insulin tolerance test ( _Kitt ,% glucose / min) according to the formula _Kitt = (0.693 / t _1/2 ) * 100, where t _1/2 is the time of the decrease in blood glucose concentration, calculated from the slope of the glucose concentration curve in the first 30 min after insulin administration [9]. For biochemical studies, blood plasma is obtained by centrifugation of whole blood (4 ° C, 8000 g, 6 min) and stored at -20 ° C for subsequent analysis. The content of high density lipoproteins, triglycerides and total cholesterol in the blood plasma of animals is determined on a biochemical analyzer (RX Imola, Randox, Japan).

The animals are removed from the experiment by CO ₂ asphyxiation. The aorta, visceral adipose tissue (retroperitoneal, epididymal, mesenteric) and liver are obtained by dissection, adipose tissue and liver are weighed on an analytical balance, and their specific gravity is calculated (tissue mass per 100 g of rat body weight). The concentration of triglycerides and cholesterol in the liver and aorta (in mg / g tissue) is determined after extraction of the lipid fraction from samples of the liver (50 mg) with a chloroform-methanol mixture (2: 1) according to J. Folch. The content of triglycerides and cholesterol in the extracted lipids is determined by enzymatic methods using kits (Chronolab, Spain). Before the analysis, a 20% solution of Thesit detergent (Sigma-Aldrich, USA) in chloroform was added to the chloroform phase. Chloroform was removed with a stream of nitrogen, the emulsified lipids were dissolved in distilled water. Working reagents from the corresponding kits were added to the resulting aqueous emulsion. The Atherogenic Index of Plasma (AIP) is calculated [10].

Statistical analysis of the data obtained is performed in the IBM SPSS Statistics 23 program. The correspondence of the obtained quantitative indicators to the normal distribution law is determined using the Shapiro-Vilk W-test. If the data obey the normal distribution law, the results are described as mean and standard deviation (M ± SD), analysis of differences between samples is performed using the Student's t-test. If the data do not obey the normal distribution law, then the results are described as the median and interquartile range (Me (Q ₁ -Q ₃ )), the analysis of differences between samples is carried out using the nonparametric Mann-Whitney U-test. Differences are considered significant at a significance level of p <0.05.

Example.

Keeping animals on a high-fat, high-carbohydrate diet for 12 weeks led to an increase in systolic and diastolic blood pressure, an increase in the specific gravity of adipose tissue and liver (Fig. 1).

An increase in animal weight is one of the typical features of MS [11,12]. In the experiment, the final body weight of the rats did not differ between the control and experimental groups (Fig. 1, Fig. 2). The explanation for the results obtained is the fact that the animals kept on the diet consumed less food rich in fat and fructose, probably due to its higher calorie content (Fig. 1). However, feeding rats with a high-fat and high-carbohydrate diet of the proposed composition led to the development of obesity. This is evidenced by an increase in the ratio of adipose tissue to body weight by more than 2 times (Fig. 1).

According to the glucose tolerance test, 30 minutes after glucose load in rats of the experimental group, the blood glucose level exceeded the level in the control group by 25.2% (p <0.05), after 60 minutes the difference was 32.4% (p <0, 05), after 2 hours the glucose level in the experimental group remained increased by 10% (p> 0.05) (Fig. 3, a). The area under the glucose concentration-time curve (AUC) in the experimental group was 809.9 ± 81.9 mmol / L * 120 min, which exceeded the value in the control group (585.5 ± 53.1 mmol / L * 120 min , p <0.05) (Fig. 3, b). 30 minutes after the injection of insulin, the difference in glucose level between the control and experimental groups was 30.1% (p <0.05), after 60 minutes - 32.5% (p <0.05), after 2 hours - 25, 7% (p <0.05) (Fig. 4, a). The area under the glucose concentration-time curve (AUC _0-120 ) in the experimental group was 440.9 ± 57.4 mmol / L × 120 min and exceeded that in the control group (307.1 ± 31.1 mmol / L * 120 min, p <0.05). The development of insulin resistance in animals of the experimental group was assessed by the change in _Kitm . It was found that K _itt in rats kept on a high-fat and high-carbohydrate diet was 1.3 ± 0.7% glucose / min, which is 36.5% lower than this indicator in animals of the control group (2.1 ± 0.7% glucose / min, p <0.05) (Fig. 4, b). Thus, the revealed hyperglycemia, impaired glucose tolerance and insulin resistance indicate the development of metabolic syndrome in animals when fed with high-fat and high-carbohydrate foods.

In a study in rats of the experimental group, an increase in blood plasma triglycerides by 2.1 times was observed compared to the control, while total cholesterol did not increase statistically significantly (Fig. 5). The development of hypertriglyceridemia is caused precisely by the lipogenic effect of fructose added to the feed [13, 14].

The level of high density lipoproteins in the blood plasma of animals receiving the developed diet did not differ significantly from the control group (Fig. 5). However, the calculated AIP in rats on a diet increased 3 times (0.2 ± 0.1 in the control and 0.6 ± 0.2 in the experimental group, respectively, p <0.05). This indicator is used in clinical medicine to assess cardiovascular risk in patients and is considered a potential biomarker for early diagnosis of cardiovascular diseases [15]. An increase in the level of triglycerides in the wall of the aorta (Fig. 5) may be a sign of developing atherogenesis. The data obtained indicate that feeding rats with a high-fat and high-fructose diet leads to the development of dyslipidemia and increases the risk of cardiovascular pathology.

There was also an increase in the specific gravity of the liver, the level of triglycerides and cholesterol in the liver in the experimental group of rats (Fig. 5). This suggests that a high-fat and high-carbohydrate diet can contribute to the development of steatohepatosis [16].

Thus, the data obtained allow us to state that the use of the proposed method for modeling metabolic syndrome using a high-fat and high-carbohydrate diet leads to the development of key signs of metabolic syndrome in animals.

The technical result is to obtain a model of metabolic syndrome, which adequately reflects the main features of metabolic disorders developing in this syndrome, based on the inclusion in the diet of animals of an increased amount of animal fats and fructose, as well as cholesterol.

The proposed method allows you to experimentally reproduce in laboratory animals pathogenetic factors and the phenomenology of disorders arising from metabolic syndrome, using a specially designed high-fat and high-carbohydrate diet containing animal fats, fructose and cholesterol. This method can be used in the development of approaches for the prevention and treatment of metabolic syndrome.

Information sources

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4. JP 2015142564 A "Model mouse of metabolic syndrome condition accompanied by no increase of weight and manufacture method of the same"

5. CN 107156049 A "Establishment method for rat model of metabolic syndrome"

6. GOST P 50258-92 Complete feed for laboratory animals. Technical conditions. Moscow: Standards Publishing House, 1992, 7 p.

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, Frohlich J. The plasnia parameter log (TG / HDL-C) as an atherogenic index: correlation with lipoprotein particle size and esterification rate in apoB-lipoprotein-depleted plasma (FER (HDL)). Clin. Biochem. 2001; 34 (7): 583-588.

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13. Lim J.S., Mietus-Snyder M., Valente A., Schwarz J.M., Lustig R.H. The role of fructose in the pathogenesis of NAFLD and the metabolic syndrome. Nat. Rev. Gastroenterol. Hepatol. 2010, 7 (5). P. 251-264.

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Figure 1. The effect of high-fat and high-carbohydrate diet on physiological parameters of rats in the control and experimental groups, M ± SD.

Figure 2. Graph of changes in body weight of animals in the control and experimental groups after 4, 8, 12 weeks of the study.

Figure 3. A graph of the dependence of the concentration of glucose in the blood of rats of the control and experimental groups at 12 weeks of the experiment (a) and the area under the curve "glucose concentration-time" (AUC) (b) in the glucose tolerance test.

Figure 4. A graph of the dependence of the concentration of glucose in the blood of rats of the control and experimental groups at 12 weeks of the experiment (a) and the rate constant of glucose utilization (b) in the insulin tolerance test.

Figure 5. The effect of a high-fat, high-carbohydrate diet on the biochemical parameters of control and experimental rats, M ± SD.

Claims (1)

A method for modeling diet-induced metabolic syndrome in laboratory animals, which consists in using a high-fat and high-carbohydrate diet, characterized in that the animals receive a diet containing standard feed (specific gravity 66%) with the addition of animal fat (rendered lard, specific weight 17%), fructose (specific gravity 17%), cholesterol (specific gravity 0.1%), and consume 20% fructose solution instead of drinking water.

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